“Musical dish” efficiently induces osteogenic differentiation of mesenchymal stem cells through music derived microstretch with variable frequency

Abstract Nonuniform microstretching (NUMS) naturally occurs in real bone tissues in vivo, but its profound effects have not been identified yet. In order to explore the biological effects of NUMS and static stretch (uniform stretch [US]) on cells, a new “musical dish” device was developed. Musical signal was used to provide NUMS to cells. More stress fibers, arranging along the long axis of cells, were formed throughout the cells under NUMS, compared with US and untreated control group, although cell morphology did not show any alteration. Whole transcriptome sequencing revealed enhanced osteogenic differentiation of cells after NUMS treatment. Cells in the NUMS group showed a higher expression of bone‐related genes, while genes related to stemness and other lineages were down‐regulated. Our results give insights into the biological effects of NUMS and US on stem cell osteogenic differentiation, suggesting beneficial effects of micromechanical stimulus for osteogenesis. The newly developed device provides a basis for the development of NUMS derived rehabilitation technology to promote bone healing.


| INTRODUCTION
Stretch, as a form of mechanical force, has been widely explored as its advanced functions in not only regulation of cell behaviors 1,2 but also injury prevention and tissue repair. 3 Various studies have explored the effects of stretch stimulation on cell differentiation by tuning different parameters such as frequency, magnitude, direction, and duration. For example, cyclic stretching with increased amplitude (2.0%, 3.5%, and 5.0%) and constant frequency (0.5 Hz) was exploited to promote osteogenic differentiation of cells. 4 Uniaxial stretching with different amplitude (8% and 12%) were performed on cells, which demonstrated that higher amplitude is required for superior expression of tenogenic genes. 5 Stretch has also been used for promoting tissue regeneration. For example, cyclic stretch (15%, 1 Hz) delivered in TGF-β1-loaded collagen scaffold enhanced tendon regeneration in both mechanical properties and organizational structure. 6 Static stretch (33%) was identified as a necessary factor for hair regeneration by regulating M2 phenotype of macrophages and activating stem cells. 7 Although many studies have been performed to reveal the effects of stretch, most of the research focused on stretch with a high magnitude (around 5%-15%), which is far from those forces observed the in vivo environment.
Stretch is nonuniform with variable frequency and magnitude in various tissues such as lung, 8 heart, 9 brain, 10 and skeletal system. Among them, stretch in bone tissues has drawn the most attention for clinical application, as bone tissues are continuously exposed to stretch stimulation during physiological activities. The structure and function of bone tissues relies on proper amount of stretch stimulation, while lack of exercises would result in the reduction of bone volume and osteoporosis. 11 In clinical application, distraction osteogenesis (DO), a surgery procedure involving cutting and separating bone and providing stretch at both ends, promotes bone repair and regeneration through stretch. For the stretch in DO, the frequency is very low (about four times per day) and the magnitude is large (over 1 mm). 12,13 In contrast, under physiological condition, bone tissues are exposed to micro-stretch with variable frequency. 6,14 However, the biological effects of such microstretch have not been identified yet.
To artificially reproduce the biophysical condition, cell-stretching devices have been widely developed. Commercial Flexercell Tension devices and two-layer microfluidic stretching system are normally used to produce static or cyclic stretch with a constant frequency. 4,6,[15][16][17] But these devices, in which stretch is caused by the deformation of the gas channels through vacuum pressure and positive air pressure, is unable to efficiently generate stretch with variable frequencies and low amplification similar to the in vivo bone condition. Advanced responsive materials, including thermal actuation relying on the phase transition of materials such as temperature responsive hydrogels 18 and liquid crystal elastomers, 19,20 magnetic actuation controlling the deformation of micropillars on a substrate with an external magnetic field, 21 and electric actuation based on the electric-mechanical coupling behavior of dielectric elastomer actuators (DEAs), have also been used to provide stretching stimulation.
Among them, cell stretchers based on DEAs exhibit performance advantages, including fast response, large deformation, optical transparency, and robust controllability. 22 Motivated by these findings, here we propose a new design of a DEA-based cell stretcher, named as "musical dish," to provide nonuniform microstretching (NUMS) to cells. A thin layer of single-wall carbon nanotube (SWCNTs) was acted as the high-voltage electrode with excellent transparency and compliance for optical cell stretching and monitoring. NUMS was applied to cells by converting a musical signal into electrical energy, followed by converting the signal further into mechanical energy via a flexible DE membrane. The effects of NUMS, US, and unstretched control groups on cell morphology, cytoskeletal organization, and osteogenic differentiation were demonstrated. Whole transcriptome sequencing was performed to identify gene expression changes. Based on these results, the "musical dish" system paves the way for the application of NUMS to direct stem cells toward osteogenic phenotypes, further offers a clinical strategy of biomechanical regulation for bone tissue repair. These findings are useful for the development of NUMS derived rehabilitation technology to promote bone healing.

| Fabrication of the musical dish
The newly developed musical dish was shown in Figure 1a

| Strain characterization of the musical dish
In the characterizations of the actuating behaviors, the radial strain was calculated through Equation (1). The results were presented as a function of both applied voltage and test number (Figure 1e,f). Due to viscoelasticity, deformations exhibited some difference between different tests. The results obtained from the first test were notably small. However, with the test number increasing, the results finally converged, which revealed that the actuating behavior of the stretcher became stable. Figure 1e,f showed a "warm-up" of six tests was essential before using the stretcher in static case.

| Establishment of musical dish culture system
The musical dish culture system consisted of a loading control system (musical signal, audio power amplifier, and voltage amplifier) and a cell culture system (musical dish) (Figure 3a). The audio power amplifier can recognize and amplify the musical signal and convert them into electric current signal. The electric current signal was transmitted to the voltage amplifier. Then, an electric field was generated in the SWCNT electrode of musical dish by connecting the fire wire electrode to high voltage and the ground wire electrode to low voltage.
The SWCNT electrode was deformed under the electric field to enlarge the area and reduce the thickness due to the attraction of heteroelectric charge (Figure 3b). slowly with the increase of the voltage, while when the voltage was greater than 3 kV, the linear strain increased significantly. When a voltage of 1 kV was applied, the SWCNT electrode produced a tensile force with the amplitude of less than 1%, which is close to the stretch range in bone tissues. 5

| Cell proliferation and deformation in the musical dish system
To investigate the feasibility of musical dish for cell growth, C3H10T1/2 cell lines were seeded on the musical dish and the Petri dish. During 5 days' observation, cell density distribution did not vary between the two groups ( Figure S1). Cell counting kit-8 (CCK-8) assay was further used to evaluate the activity of the cells cultured on the musical dish and Petri dish at Days 1, 3, and 5, respectively. The results confirmed that no significant difference in the CCK-8 assay was observed between the two groups ( Figure 3d), suggesting no mechanical cell injury or breakage in the self-made musical dish.
We then examined whether the deformation of the SWCNT electrode of musical dish can indeed have an impact on cells. Cells seeded on the musical dish were labeled with DAPI (nucleus) and TRITC (cytoskeleton). A voltage of 0, 1, 2, and 3 kV was applied.
The results showed that when the system was loaded with a constant voltage, the SWCNT electrode stretched, leading to the dislocation of the nuclear position and the disturbance of the

| The effect of stretch stimulation on cell morphology and cell cytoskeleton
To evaluate the effect of NUMS on cells, 1 kV constant voltage with musical signal was applied to cells ( Figure 4a). According to the equation y = 1.7x-0.85 (Figure 3c), a variable stretch was estimated to range from 0.833% to 0.867% (48.73-50.71 μm) along with musical frequency in the NUMS group. The frequency of the musical signal ranged between 0 and 20,000 HZ and mostly below 1000 ( Figure 4b).
In contrast, in the US group, cells are subjected to a constant stretch of 0.850% (49.73 μm) when 1 kV voltage was applied without musical signal input. Cells cultured on the musical dish without any stretch stimulation served as a control group.
After the stimulation for 4 days, cell morphology of the NUMS group and US group was similar with the control group. Cell arrays were nondirectional. Cell protrusions became shorter, and the cytoplasm and nucleus gradually enlarged ( Figure S2). Quantitative analysis showed that there was no significant difference in cell area and aspect ratio after stimulation in the NUMS and US groups, compared with the control group (Figure 4c To gain further functional insights, the gene ontology (GO) enrichment analysis was performed. The top 20 significantly enriched items of GO terms between the NUMS and control group included triglyceride catabolic process, neutral lipid catabolic process, acylglycerol catabolic process, pyruvate metabolic process, ADP metabolic process, glycolytic process ( Figure S4A). The GO terms between the NUMS and US group were significantly enriched in chromatinmediated maintenance of transcription, cellular response to vitamin D, necroptotic process, plasma lipoprotein particle assembly, proteinlipid complex assembly, regulation of platelet-derived growth factor receptor signaling pathway ( Figure S4B).
We then focused on the GO terms related to cell fate regulation.

| The effect of stretch stimulation on cell differentiation
To further investigate cell fate determination, genes related to cell fate were tested after stimulation for 9 days. The results showed that the expression of stemness-related genes Sox2 and Nanog decreased significantly in the NUMS and US groups than that in the control group (Figure 6a (Figure 6f,g). The results of alizarin red staining showed typical mineralized nodules were formed in the NUMS group, while atypical mineralized nodules were formed in both the US and control groups (Figure 6h). Quantitative results showed that the NUMS group had significantly higher alizarin red staining compared to the US group and control groups ( Figure 6I). These results further demonstrated that the NUMS can promote osteogenic differentiation of cells.

| CONCLUSION
Our findings highlight a newly developed device, "musical dish," which allows arbitrary strain waveforms to be applied to cells by converting music signal into stretching force (Figures 1a,b and 3a). A bionic NUMS was subjected to cells through the musical dish (Figure 4a,b).

| DISCUSSION
The reason for the lack of significant difference in cell morphology between the groups could be due to the small stretch amplitude in the NUMS and US groups. The effect of stretch on morphological alteration depends on strain amplitude, frequency, and duration. 25 Thresholds of these parameters that can affect cell morphology vary in different studies. For example, cells exposed to a stretch treatment with an amplitude of 10% and a frequency of 1 Hz showed similar shape index with the control group without any stretching. 26 Under 5% stretching, the aspect ratio of the cells showed only a small change compared with the control group without stretch. 27 Another study showed that stretch with 5% amplitude and 1 Hz frequency did not change the morphology of cells. 28 It has been reported that even if cells were stimulated by 1% and 2.75 Hz cyclic stretch, they showed similar area and shape index with the control group without stretch. 29 Here, the stretch amplitude of the NUMS and US is 0.85%. Thus, we believe that the aspect ratio will not change significantly under the stretch induced by such a low amplitude.
Although cell morphology is closely related to cell fate, changes in cell morphology are not a necessary for cell fate regulation. For example, cells under 10% and 1 Hz stretch showed similar morphology with the untreated group, but the stretch induced tenogenic differentiation of the cells. 26 Another study found that when cells encapsulated in hydrogels with different stiffness showed similar morphology, cells fate can also be regulated by the stiffness. 30 Previous studies have found that, in addition to maintaining cell morphology, cytoskeleton participated in the transmission of mechanical signals, which changed the activation, synthesis, secretion, and other functions of corresponding effector molecules, thus affecting cell proliferation, differentiation, movement, and other activities. 31,32 In addition, the cytoskeleton links to Lamin A through the LINC complex to anchor the chromatin. Therefore, cytoskeletal tension has the potential to directly alter gene expression. 33 The GO analysis results also showed that neural differentiation was enriched in the NUMS group compared with the US group (Figure 5f).
There is accumulating evidence suggesting "neuro" related processes can shed light on "osteo" related processes. For examples, Runx2 is functionally connected to many genes that are important for brain and language development, but also to bone formation. 34 Runx2 directly interacts with Satb2, a gene that regulates stereotypic projections in the cortex. 35 This gene also plays a key role in osteoblast differentiation. 36 Moreover, Runx2 interacts with Dyrk1a, a gene that not only controls neural precursor activity and differentiation but also involves in bone homeostasis as an inhibitor of osteoclastogenesis. 37,38 Other neurogenetic factors such as Cbl, Foxp2, Hes1, and Akt1 were also reported to interact with Runx2. [39][40][41][42] Based on the crosstalk between the osteogenic and neurogenic stem cells, we believe that the NUMS may have great potential to promote bone differentiation of cells.
For clinical application, three strategies may contribute to the development of potential therapeutic ways for bone regeneration.
First, the NUMS could be used in the DO surgery to replace or assist the current large-scale and slow stretching method for bone regeneration due to its excellent performance in promoting osteogenic differentiation. The second is the "musical dish," which could be further developed into a music derived rehabilitation technology to promote bone healing. Finally, the musical dish can serve as a cell expansion bioreactor to produce cells with high level of osteogenic differentiation to repair bone repair by transplantation. Thus, both the new stretch model and the newly developed device for stretching may have a great potential for clinical application. and (3) transferring the percolation mat onto the prestretched VHB membrane. After the SWCNT electrode was transferred to VHB membrane, the VHB membrane was sandwiched by two PMMA rings to form a DEA stretcher. The fabricated DEA stretcher was fixed to a hollowedout dish (Corning, USA) to form the musical dish. Thin strips of tinfoil were used as conductive tape for the cables of the high-voltage power supply. Graphite carbon paste was used to guarantee the tight contact between the SWCNT electrode and the tinfoil. At last, to protect the SWCNT electrode, a thin layer (about 50 μm) of polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) was spin coated at 4000 rpm. To reduce the stiffening effect of the PDMS to the VHB membrane, a 20:1 monomer-curing agent ratio was adopted. The temperature and time for curing were set to be 40 C and 12 h.

| Strain characterization of the musical dish
The actuating strains of the stretcher under different applied voltages were measured. Potable natural mineral water (NONGFU SPRING) was used as the ground electrode. The voltages (3, 4, and 5 kV) were generated by a high-voltage power supply (610E, TREK, USA). A DSLR camera (70d, Cannon, Japan) was used to capture the profiles of the SWCNT electrode during actuation. Considering the viscoelasticity of VHB membrane, each picture was shot 1 min after the onset of the voltage. And before a new voltage was applied, there was 1 min for the stretcher to recover to its initial state. The radial strain was then calculated as: where r 0 is the initial radius of the electrode and Δr is the difference of the radiuses before and after the voltage was applied. The radius of the electrode during actuating was measured as half the distance between point A and point B, as shown in Figure 1d. The procedure was repeated nine times and there was 10 min for relaxation each time after the voltage is off.
To assess the actuating stability, two square-wave driving signals at 0.15 Hz and 1 Hz with a 50% duty cycle were generated respectively by a function signal generator (AFG1022; Tektronix, USA) which was connected to the high-voltage power supply (610E, TREK). The stretcher was then cycled between 0 and 5 kV for 4 h. At specific time points (0, 10, 30, 120, and 240 min), a 1 min period of video was shot to record the dynamic response of the stretcher. The radial strain, calculated by Equation (1), was taken as the average value in that minute.

| Model analysis of the homogeneity and equiaxiality of the musical dish
The active part (electrode covered area, including the central circular zone and the electrode lead zone) shrinked in thickness under the Maxwell stress: where ε 0 is the permittivity of vacuum, p r is the dielectric constant of the elastomer, E is the electric field, V is the applied voltage, and t is the thickness of the DE membrane.
Commercially available finite element analysis (FEA) package ABAQUS was used to obtain the strain distribution of the active part of the VHB membrane. The geometric parameters of the model were set to be the values before prestretch. Yeoh model was used to describe VHB's hyperelasticity and incompressibility. The Yeoh form of strain energy potential was described as: where I 1 is the first invariant of the left Cauchy-Green deformation tensor and C 10 , C 20 , C 30 were three material parameters. The material parameters of VHB 4910 were listed in Table 1. 44 The modeling process was composed of two steps. In the first step, a displacement was applied to stretch the membrane by 200% radially. In the second step, a pressure was imposed to squeeze the active part in thickness. The amplitude of the pressure (with the applied voltage of 5 kV) was calculated by Equation (2). This approximation was well suited for planar actuators. 44

| Linear strain of musical dish
Linear strain was measured by the same method as "radial strain" to describe the stretchability of the musical dish. Cell culture medium was used as the ground electrode. A series of constant voltages of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 kV were applied. The linear strain (w%) was calculated by a formula: where ε r is the radial strain.

| Cell culture and proliferation
The mouse cell line C3H10T1/2 was obtained from Cell Bank of the on Illumina X-Ten platform. Sequence reads were mapped to reference genome mm10 using Bowtie2 using default parameters and per gene counts were calculated using HTSeq. R statistical programming language was utilized to analyze all statistical data. DESeq2 was used to identify differentially expressed genes (DEGs). In our evaluation, a gene was considered to be expressed in a sample if its count value was equal or greater than 1 in the sample. DEGs were illustrated as fold-change ≥2 and p value ≤ 0.05. GO analysis was performed using DAVID (http://david.ncifcrf.gov).

| Quantitative RT-PCR analysis
The total RNA was extracted by TRIzoL rapid extraction method as above. RNA was reverse transcribed using 5 Â RT Master Mix (TOYOBO, Japan) and 2 μg RNA template. The amplification procedure was used as follow: 37 C for 15 min; 50 C for 5 min and 98 C for 5 min in PCR Amplifier (Biometra, Germany). RT-PCR was performed using 25 ng of cDNA samples using 5 mmol/L of each primer (

| Statistical analysis
In this experiment, all data were expressed as mean standard deviation, and their statistical significance was evaluated by t-test. The ttest was carried out with the function of T.TEST in Microsoft Excel.